Characterization of the complete chloroplast genome of Carallia brachiata (Lour.) Merr. (Rhizophoraceae)

Abstract Carallia brachiata (Lour.) Merr. (1919) is an important medical resource distributed across subtropical Asia. In this study, the complete chloroplast genome of C. brachiata was sequenced, revealing a total length of 162,460 bp, including four regions – a large single copy (89,814 bp), a small single copy (18,804 bp), and a pair of inverted repeats (26,921 bp each). The overall guanine + cytosine content was 35.76%. In total, 130 genes were annotated within the chloroplast genome, comprising 85 protein-coding, 37 tRNA, and 8 rRNA genes. Subsequent phylogenetic analyses revealed that C. brachiata is closely related to Carallia diplopetala.


Introduction
Carallia brachiata (Lour.) Merr. (1919) is a member of the genus Carallia in the family Rhizophoraceae and is mostly distributed across subtropical Asia. Its leaves are oval and have smooth surfaces and edges ( Figure 1). It is an important medical resource for treating sapraemia, and its bark is used in pruritis treatment (Ling et al. 2004). However, there is no record of the complete chloroplast genome of C. brachiata in the National Center for Biotechnology Information (NCBI) database. Therefore, in this study, the complete chloroplast genome of C. brachiata was sequenced, and its phylogenetic position within the Rhizophoraceae was confirmed.

Materials and methods
Fresh leaves of C. brachiata were collected from the South China Botanical Garden in Guangzhou,China (23 11'16.8" N,113 22'15.6 00 E) in compliance with the national Wild Plant Protective Regulations. The Biomarker Technologies Corporation (Beijing, China) approved the collection of the required samples for analysis. A specimen was deposited at Biomarker Technologies Corporation (Jian Zhao, email: zhaojian0102@outlook.com) under the voucher number ZJS202101110ZJ. The total genomic DNA was extracted from the fresh leaves using the modified CTAB method (Doyle and Doyle 1987), and libraries were prepared using the NexteraXT DNA Library Preparation Kit (Illumina, San Diego, CA). The libraries were then sequenced on the Illumina Novaseq 6000   platform, and the raw data obtained were filtered using PRINSEQlite v. 0.20.4 (Schmieder and Edwards 2011), yielding 3.46Gb of clean data with a read coverage depth over 600X ( Figure 2). High-quality reads were assembled into the chloroplast genome using de novo assembler SPAdes v.3.11.0 (Bankevich et al. 2012). Finally, the complete chloroplast genome was annotated using the PGA software package (Qu et al. 2019), with the chloroplast genome of Pellacalyx yunnanensis (MN106253) serving as a reference. The results were then submitted to GenBank under accession number OM141003. Sixty-two homologous protein-coding genes (PCGs) from 26 chloroplast genomes in the NCBI were selected using OrthoFinder v2.3.14 (Emms and Kelly 2015). These were aligned with the C. brachiata genome using MUSCLE v.3.8.1551 (Edgar 2004), and conserved sequences were extracted from the alignment using Gblocks v0.91b (Talavera and Castresana 2007). Prottest v3.4 was used to select the HIVb þ I þ G þ F model, and Couratari macrosperma (MF359944.1) from Lecythidaceae was used as the outgroup. Finally, IQtree v. 1.6 was used to construct a maximum likelihood tree with 1000Â bootstrap resampling (Nguyen et al. 2015).
Twenty-nine species were initially used to construct the phylogenetic tree; however, the bootstrap value was too low to be valid; thus, the related species were removed. As a result, the final phylogenetic tree consisted of 27 species. The phylogenetic analysis revealed that C. brachiata was more closely related to Carallia diplopetala among all members of the Rhizophoraceae family (Figure 4).

Discussion and conclusion
In this study, the complete chloroplast genome of C. brachiata was sequenced, revealing a total length of 162,460 bp, including four regions: a large single copy (89,814 bp), a small single copy (18,804 bp) and a pair of inverted repeats (26,921 bp each). The overall GC content was 35.76%. In total, 130 genes were annotated within the chloroplast genome, including 85 PCGs and 37 tRNA and 8 rRNA genes. Subsequent phylogenetic analyses revealed that C. brachiata is closely related to Carallia diplopetala (NC_062600.1). C. diplopetala, which exhibits the closest relationship to C. brachiata, has a slightly smaller chloroplast genome than C. brachiata, comprising 83 PCGs, 37 tRNAs and 8 rRNAs, with a total length of 162,052 bp (Wang et al. 2021).

Author contributions
You Zhou and Xiongmei Zhu performed the experiments, analyzed the data, authored drafts of the paper, and approved the final draft. Jiyun She analyzed the data, prepared the figures, and approved the final draft. Fen Xiao and Jian Zhao conceived and designed the experiment, reviewed the drafts of the paper, and approved the final draft. All authors agree to be accountable for all aspects of this study.

Ethical statement
Carallia brachiata leaves were collected from the South China Botanical Garden in Guangzhou, China in compliance with the national Wild Plant Protective Regulations.

Disclosure statement
No potential conflict of interest was reported by the author(s).